Stem cell-based therapies, including the recent CD34+ cell therapy, are a promising therapeutic approach for improving cardiac regeneration and function. The benefits of CD34+ cell transplantation appear to occur primarily via increase in vascular angiogenesis by the CD34+ cell-secreted paracrine factors in the ischemic myocardium. However, the precise mechanisms that lead to CD34+ cell-induced vessel growth and therapeutic recovery are poorly understood. This lack of mechanistic insight is a critical barrier to the success of cardiac stem cell therapy, as the regenerative efficacy of the transplanted stem cells is limited by their poor viability and retention in the ischemic myocardium. Therefore, to address these limitations and to develop novel alternate approaches, we must seek to understand the mechanisms that lead to therapeutic recovery. Our previously published data and our preliminary data have revealed that the paracrine secretion from human CD34+ cells contain membrane-bound nano-vesicles called exosomes (i.e. CD34+ exosomes), which are angiogenic and therapeutic similar to the cells. Further, CD34+ exosomes carry and transfer proangiogenic miRNAs, such as miR-126, to ischemic endothelial cells in the myocardium and induce their angiogenic activity. The fundamental basis of our proposal is to harness the regenerative potential and communication power of CD34+ exosomes to augment therapeutic approaches. Our central hypothesis is that exosomes released via paracrine secretion of human CD34+ cells mediate myocardial repair by direct transfer of miRNAs to cells in the heart. Our goal is to establish CD34+ stem cell-derived exosomes as a novel cell-free therapeutic entity for ischemic myocardial repair and to develop a comprehensive understanding of the molecular mechanisms of exosomes-induced cardiac angiogenesis and therapeutic recovery. This innovative study is important to understand the mechanisms of CD34+ cell therapy and to unlock the transformative potential of progenitor cell-derived exosomes. We will test our hypothesis in three specific aims: 1) Determine the therapeutic efficacy of cell-free CD34+ exosomes in a murine model of myocardial ischemia and determine whether CD34+ exosomes secretion is one of the key mechanisms of CD34+ cell therapy; 2) Characterize the molecular mechanisms of CD34+ exosomes-induced angiogenesis by studying the CD34+ exosomes trafficking and signaling mechanisms in the ischemic heart; 3) Establish the extent to which the beneficial effects of CD34+ exosomes are mediated by miR-126 in a murine model of MI. The experiments described in this proposal will explore key scientific questions by characterizing the predominant, but as yet undefined, mechanism of CD34+ stem cell therapy. Our studies will lay foundation to a novel therapeutic approach by using exosomes from human stem cells as a suitable cell-free alternative. It has the potential to advance cell- based therapis by exploiting many practical and technical advantages of exosomes relative to cells for application in cardiovascular regenerative medicine.